The nature of protein metabolism in Bacillus subtilis during sporulation is to be studied. Growing cells will be continuously labeled with 14C-leucine and transferred to medium containing 3H-leucine during sporulation. The total soluble proteins present in the cell during sporulation will be analyzed; this analysis will be accomplished by doing isoelectric focusing in poly-aerylamide in one direction followed by electrophoresis in sodium dodecylsulfate-polyacrylamide gel in the second direction. Absolute counts and ratios of counts will be determined by direct scintillation counting and by autoradiography. It is hoped to learn whether, as in mammals, a given protein undergoes simultaneous synthesis and degradation. Using the methods developed over the last two years, it is proposed to prepare sufficient intracellular metalloserine protease to prepare specific antibodies. The antibodies are to be used to verify the time of appearance of this protease in the cell during growth and to attempt to determine its intracellular location. Syntheses of specific aldehyde and sulfonamide serine protease inhibitors are to be attempted. These will be used to assess the roles of the sensitive serine protease in protein turnover and sporulation. Three methods are proposed for isolation of intracellular protease-deficient mutants: 1) screen for inability to hydrolyze a specific chromogenic substrate, 2) screen for conditional requirements of amino acids for sporulation and 3) screen for resistance to selective protease inhibitors. The ability of any such mutants to carry out protein degradation and form spores will be studied.